Both chemotherapy and radioimmunotherapy induce dose-limiting myelosuppression. In fact, chemotherapy-induced myelosuppression is the most common dose-limiting, and potentially fatal, complication of cancer treatment. Maxwell et al., Semin. Oncol. Nurs. 8:113–123 (1992); Blijham, Anticancer Drugs 4:527–533 (1993). Drug-induced hematopoietic toxicity is a common reason for curtailing high dose chemotherapy in cancer patients (Boesen et al., Biotherapy 6:291–302 (1993)), and higher dose chemotherapy is only possible in conjunction with bone marrow transplantation (BMT), autologous stem cell infusion, and treatment with hematopoietic growth factors.
During the recovery period after anticancer myelosuppressive therapy, hematopoietic progenitor cells become mitotically active in order to replenish the marrow compartment and remain hyperproliferative even after normalization of peripheral white blood cells (pWBCs) and platelets (PLTs). At this stage, the progenitors are more radio- and chemo-sensitive. Dosing patients with additional cytotoxic therapy during this phase will likely result in more severe toxicity.
As a general model of myelosuppressive therapy, acute damage and recovery of hematopoietic stem and precursor cells following whole-body irradiation also has been studied extensively. Testa et al., Anticancer Res. 5:101–110 (1985); Sado et al., Int. J. Radiat Biol 53:177–187 (1988); Meijne et al, Exp. Hematol. 19:617–623 (1991). External beam irradiation results in long-term damage of hematopoietic stem cells, which manifests with the presence, but at sub-optimal levels, of mitotically active, hematopoietic progenitor cells (CFU-S) 3–6 months after treatment. Lorimore et al., Int. J. Radiat Biol 57:385–393 (1990); Lord et al., Int. J. Radat. Biol. 59:211–218 (1991). Persistent depletion of femoral and splenic CFU-S (colony forming unit-spleen), CFU-GM (colony forming unit-granulocytic-monocytic) and BFU-E (burst forming unit-erythroid) can occur, even though the peripheral blood contains normal cell numbers. Grande et al., Int. J. Radiat. Biol. 59:59–67 (1993). Severe reduction in the supportive stroma has also been reported. Tavassoli et al., Exp. Hematol. 10:435–443 (1992). Following radiation exposure, recovery proceeds by repair of sublethal cellular injury and compensatory cellular repopulation by the surviving fraction. Hall in RADIOBIOLOGY FOR THE RADIOBIOLOGIST (Harper & Row 1978); Jones et al., Radiation Res. 128:256–266 (1991).
Normal white blood cell (WBC; >4000/mm3) and platelet (PLT; >100,000/mm3) counts are the usual markers for patient tolerance to repetitive myelosuppressive treatment. However, preclinical and clinical evidence suggests that peripheral counts are not a reliable surrogate for predicting complete myelosuppressive recovery. Although WBC and PLT counts may appear normal, the primitive stem and progenitor cell compartments are not fully recovered from previous myelosuppressive therapy.
Further cytotoxic treatment while stem cells and progenitor cells are rapidly proliferating can result in more severe myelotoxicity or even death. One solution to this problem is to collect bone marrow (BM) aspirates and use a long-term culture system to quantitate high proliferative potential CFC (HPP-CFC) or long term culture initiating cells (LTC-IC). Eaves et al., Tiss. Culture Meth. 13:55–62 (1991); McNiece et al., Blood 75:609–612 (1989). While this method can provide the needed information, such assays take 3–6 weeks to perform, and thus are not clinically useful.
During hematopoiesis, pluripotent stem cells differentiate and proliferate in multiple lineages. The process proceeds under the permissive influence of “early” and “late” hematopoietic cytokines. Lowry et al., J. Cell Biochem. 58:410–415 (1995). “Early” stimulatory factors include SCF, FLT-3-L, IL-1, IL-3, IL-6, and IL-11. In addition to these positive regulators, hematopoiesis is also controlled by inhibitory cytokines. Negative regulation of myelopoiesis occurs through several inhibitory cytokines, most notably MIP-1α (Cooper et al., Expt. Hematol. 22:186–193 (1994); Dunlop et al., Blood 79:2221–2225 (1992)), TGFβ3 (Jacobsen et al., Blood 78:2239–2247 (1991); Maze et al., J. Immunol. 149:1004–1009 (1992)) and TNFα (Mayani et al., Eur. J. Haematol. 49:225–233 (1992)).
Thus far a temporal change in these inhibitory peptides as a function of time after cytotoxic therapy has not been quantitated. It is known, however, that under stressful conditions, such as irradiation, chemotherapy, blood loss, infection or inflammation, both stimulatory and inhibitory growth factors play a major role in cellular adaptation processes. Cannistra et al., Semin. Hematol. 25:173–188 (1988). Under stress, the quiescent CFU-S component of the stem cell compartment is triggered into active cell cycling and returns to the predominantly G0G1 phase once normal bone marrow cellularity is restored. Becker et al., Blood 26:296–304 (1965).
The recent literature has highlighted several important areas where a noninvasive method to monitor myelorecovery could have considerable clinical benefit. For example, to improve the safety and cost effectiveness of high-dose regimens, hematopoietic cell support (cytokines) has been used to accelerate marrow recovery following myeloablative therapy. This approach results in an earlier recovery of peripheral blood counts, but the proliferative status of the marrow remains unknown and could be in a very active and sensitive state.
Another relevant example pertains to the use of allogeneic or autologous BMT, or more recently peripheral stem cell transplantation (SCT) following myelosuppressive or myeloablative therapy. Under those conditions, hematopoiesis is characterized by a prolonged and severe deficiency of marrow progenitors for several years, especially of the erythroid and megakaryocyte types, while the peripheral WBCs and PLTs have reached relatively normal values within a few weeks. Therefore, successful engraftment can not be measured by normalization of WBCs or PLTs, but requires another type of marker, perhaps one associated with normal marrow stromal function. Domensch et al., Blood 85:3320–3327 (1995). More information is needed to determine ‘true’ myelorecovery when either BMT or SCT is utilized. Talmadge et al., Bone Marrow Transplant. 19(2):161–172 (1997).
Yet, another area where a noninvasive measure of myelorecovery may be useful is for scheduling leukapheresis. Since patient-to-patient variability in time to marrow recovery is quite variable following G-CSF stem ell mobilization, it is difficult to predict the best time for this procedure. Identification of one or more markers of myelotoxic nadir and recovery could advance SCT technology. Shpall et al., Cancer Treat. Res. 77:143–157 (1997).
One investigator has shown that after allogeneic or autologous BMT, a rise in endogenous G-CSF levels precedes and correlates with myeloid engraftment. Cairo et al., Blood 79(7):1869–1873 (1992). Moreover, in patients suffering from acute bacterial infections, whose rate of myelopoiesis must adapt to the enhanced demand, G-CSF, but not GM-CSF, was elevated. Selig et al., Blood 79:1869–1873 (1995). Additional studies demonstrated that the stem cell subset responsible for reconstitution is responsive to GM-CSF, IL-3, IL-6, and SCF. Wagemaker et al., Stem Cells 13:165–171 (1995). Other reports have quantified one or more cytokines during a myelosuppressive episode. Sallerfors et al., Br. J. Hematol. 78:343–351 (1991); Baiocchi et al., Cancer Research 51:1 297–1303 (1996); Chen et al., Jap. J. Clin. Oncol. 26:18–23 (1996). Heretofore, however, no one carefully studied the recovery phase following myelosuppression, and there exists no correlation with the ability to redose without severe toxicity. A relatively new stromal cell-produced positive stimulatory cytokine, FLT-3-L (Brasel et al., Blood 88:2004–2012 (1996); Lisovsky et al., Blood 88(10):3987–97 (1996)), has not been studied at all to date regarding either constitutive or induced hematopoiesis. The ability to predict the magnitude of myelotoxicity in response to a given dose of RAIT would permit patient-specific dosing. Red marrow absorbed doses have not been highly predictive of hematopoietic toxicity in RAIT-treated patients. DeNardo G L, DeNardo S J, Macey D J, Shen S, Kroger L A. Overview of radiation myelotoxicity secondary to radioimmunotherapy using 131I-Lym-1 as a model. Cancer. 1994; 73:1038–1048. Juweid M E, Zhang C, Blumenthal R D, Hajjar G, Sharkey R M, Goldenberg D M. Prediction of hematologic toxicity after radioimmunotherapy with 131I-labeled anticarcinoembryonic antigen monoclonal antibodies. J Nucl Med. 1999; 40:1609–1616.
Although the dose-toxicity relationship is likely to improve as more patient-specific models for the calculation of red marrow dose are implemented, more work needs to be done to define the tolerance of patients who have received therapy prior to nonmyeloablative radioimmunotherapy (RAIT). Thus, methods need to be established that reflect more accurately the marrow reserve in patients, so that the activity prescription for RAIT can be adjusted accordingly.
In previous work (Blumenthal R D, Lew W, Juweid M, Alisauskas R, Ying Z, Goldenberg D M. Plasma FLT3-L levels predict bone marrow recovery from myelosuppressive therapy. Cancer. 2000; 88:333–343), it was demonstrated that 13% of the patient population studied experienced significantly less toxicity than was predicted by their marrow dose and 15% of the same population experienced significantly greater toxicity than predicted. Many of these patients have received multiple treatments of external beam radiation therapy and/or chemotherapy prior to receiving RAIT. It was postulated that long-term hematopoietic damage from prior cytotoxic therapy might render a patient's marrow more “briftle” and therefore more radiosensitive to the RAIT dose. Additional tumor-produced cytokines may also be a significant factor influencing the proliferation rate of marrow cells, thereby affecting their response to radiation from RAIT. R. D. Blumenthal, A. Reising, E. Leon, and D. M. Goldenberg. Modulation of marrow proliferation and chemosensitivity by tumor-produced cytokines from syngeneic pancreatic tumor lines. American Society of Hematology Annual Meeting Abstracts, 2001; #946.
FLT3-L is a growth factor involved in early hematopoiesis, is expressed in transmembrane and soluble forms, and stimulates/co-stimulates proliferation and colony formation of hematopoietic myeloid and lymphoid stem/progenitor cells (CFU-GM and CFU-GEMM) in bone marrow, spleen and peripheral blood. Lisovsky M, Braun S E, Ge Y, et al. Flt3-ligand production by human bone marrow stromal cells. Leukemia. 1996; 10:1012–1018. Brasel K, McKenna H J, Morrissey P J, et al. Hematological effects of flt3-Ligand in vivo in mice. Blood. 1996; 88:2004–2012. Papayannopoulou T, Nakamoto B, Andrews R G, et al. In vivo effects of flt3/flk2-ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor. Blood. 1997; 90:620–629. By itself, FLT3-L has weak colony-stimulating activity, but is additive to greater-than-additive on colony number and size when combined with other colony stimulating factors (CSFs). In addition, a need still exists to establish a predictive marker for a sizeable number of individuals who experience significantly less toxicity for a given marrow dose of RAIT, than was expected.
Therefore, a need exists in the art for improved methods, and kits for implementing them, for predicting myelosuppressive recovery in conjunction with the foregoing deficient therapeutic techniques. Such methods could be used to help optimize treatment, informing the clinician of the appropriate timing of treatment, especially retreatment, thus avoiding toxic effects, while maximizing efficacious ones. Provided such a method, the art would posses new, optimized methods of treatment.